Pyruvate: ferredoxin oxidoreductase (PFOR) is an essential enzyme of central metabolism in all strictly anaerobic bacteria, anaerobic human parasites and in epsilon proteobacteria (Helicobacter and Campylobacter). Amixicile, a first-in-class amino-nitrothiazole propylamine antibiotic developed by our team, specifically inhibits PFOR and related enzymes, where both mechanistic enzymology and modeling studies suggest a chemical inhibition mechanism involving deprotonation of the activated thiamine pyrophosphate (TPP, a derivative of vitamin B1) cofactor, thus inactivating the PFOR catalytic cycle1. Significance: This mechanism appears to escape mutation based drug resistance as chemical changes to vitamins like TPP are believed to be lethal. Importantly, resistance to amixicile has not been observed with Clostridium difficile clinical isolates or through in vitro mutant generation methods with H. pylori. Amixicile is not redox-active, mutagenic or a substrate of nitroreductases; and demonstrates greater target selectivity, bioavailability and lower toxicity compared to parent FDA-approved drug nitazoxanide (NTZ). Amixicile was developed to treat C. difficile infections (CDI) and in a mouse CDI model, considered predictive of human efficacy, amixicile showed superiority to NTZ and equivalence to standard therapies (fidaxomicin and vancomycin) at 5 days and superiority over both drugs by day 14 with no recurrence of CDI. Amixicile does not harm beneficial probiotic microflora. Amixicile has completed preclinical evaluation with excellent pharmacological metrics (ADME, toxicology and PK) and is on track for phase 1 clinical trials. The proposed studies will validate the PFOR drug target and assess therapeutic efficacy of amixicile against Cryptosporidium parvum, Giardia lamblia, Trichomonas vaginalis, Entamoeba histolytica and Helicobacter pylori to meet the feasibility or use in new interventions objective, and for lead optimization and screens to evolve more potent analogues. We have assembled a collaborative team of experts in medicinal chemistry, parasitology, microbial biochemistry and animal models and have developed a pipeline approach to fast track second generation leads as outlined below in the specific aims for R21 and R33 enabling studies. The R21 phase of these studies will include: Aim 1 to evaluate amixicile in mouse models of infection for Cryptosporidium parvum, Giardia lamblia, Entamoeba histolytica, and Helicobacter pylori; Aim 2 performs a medicinal chemistry directed lead optimization of amixicile by structure activity relationships and begins exploring coupling of amino nitro-thiazole to existing antibiotics based on the success of creating ciprothiazole from ciprofloxacin and aim 3 will complete the R33 portion of the studies by in vitro and in vivo testin of developed leads from amixicile and novel multi- target therapeutics created in Aim 2. Efficacy studies and preliminary PK studies with new analogues will be prioritized for further preclinical evaluation. Our strategy of developing new therapeutics that target vitamin cofactors and multiple drug targets is designed to overcome typical pathways of drug resistance.